The promise of lentiviral gene transfer is currently limited by questions relating to safety concerns. To address this issue, Project 2 will complement Project 1 by characterizing integration by lentiviral vectors in patient cells. We have previously cloned and sequenced over 4000 integration sites made by retroviral infection of human cells and mapped them on the draft human genome sequence, providing key background for analyzing integration during gene therapy trials. We find that lentiviral integration strongly favors genes and gene-rich regions, and that active genes are particularly favored. Other retroviruses such as MLV and ASLV show different target site preferences. In collaboration with Dr. June and colleagues, we have begun analyzing integration sites generated from cells obtained from HIV infected patients enrolled on the first human gene therapy protocol employing lentiviral vectors. The numbers of integration sites analyzed so far is low (210), but the data do indicate preferential integration in active genes by these vectors as well. We propose to carry out a comprehensive study of integration in patients generated during Project 1. We will determine the locations of a much larger number of integration events in patients and study the distribution of sites in the human genome. The hazards of integration will be addressed by quantifying the frequency of integration in undesirable targets such as proto-oncogenes or tumor suppressors. Integration sites will also be analyzed longitudinally in patients after reintroduction of transduced cells, allowing any influence of integration site placement on cellular persistence in vivo to be detected. We will optimize methods for cloning integration sites from small amounts of material, allowing analysis of integration sites in diverse patient tissues where input DNA is limiting. Lastly, we will begin to examine treatments of the cellular population that have the potential to allow some measure of control of integration site selection. In our first experiment we will analyze integration site selection in resting T-cells to determine whether this results in increased integration outside genes. Our Specific Aims are as follows: (1) Characterize integration site sequences of lentiviral vectors in patient samples; (2) Characterize integration site patterns using EPTS/LM-PCR; (3) Develop methods for cloning integration sites from low abundance materials, and (4) Analyze HIV integration targeting in resting T-cells, to test the specific hypothesis that diminished gene activity in resting T-cells results in a decreased frequency of integration in genes, with the eventual goal of promoting integration outside of genes.